The present invention relates to the field of manufacturing food products by means of lactic acid bacterial cultures. Specifically the invention provides novel genetically modified strains of lactic acid bacteria that are modified to have enhanced or reduced diacetyl reductase activity, acetoin reductase activity and/or butanediol dehydrogenase activity. Such modified bacteria are particularly useful in the manufacturing of food products having either a reduced or an increased content of the flavour compound diacetyl.
Lactic acid bacteria are used extensively as starter cultures in the food industry in the manufacturing of fermented products including milk products such as e.g. yoghurt and cheese, meat products, bakery products, wine and vegetable products. Lactococcus species including Lactococcus latcis are among the most commonly used lactic acid bacteria in dairy starter cultures. Several other lactic acid bacteria such as Leuconostoc species, Pidococcus species. Lactobacillus species, Oenococcus species and Streptococcus species are also commonly used in food cultures.
When a lactic acid bacterial starter culture is added to milk or any other food product starting material under appropriate conditions, the bacteria grow rapidly with concomitant conversion of citrate, lactose or other sugar compounds into lactic acid/lactate and possibly other acids including acetate, resulting in a pH decrease. In addition, several other metabolites are produced during the growth of lactic acid bacteria. These metabolites include ethanol, formate, acetaldehyde, xcex1-acetolactate, acetoin, diacetyl, carbon dioxide and 2,3 butylene glycol (butanediol).
Among these metabolites, diecetyl (2,3-butanedione) is an essential flavour compound in dairy products such as butter, yoghurt, starter distilate, margarine, buttermilk and cheese. However, its presence in other products, such as fruit juices, beers and wines, is undesirable, as it imparts a buttery or toffee taste and is the agent responsible for the so-called sarcina sickness of beer. The compound is formed during fermentation of lactic acid bacterial species of e.g. Lactococcus, Leuconostc and Lactobacillus by an oxidative decarboxylation of xcex1-acetolactate which is formed from two molecules of pyruvate by the action of xcex1-acetolactate synthase (ALS)
Diacetyl reducing enzymes, commonly termed diacetyl reducteses (DR) (acetoin:NAD oxidoreductases E. C. 1.1.1.5), have been observed from many different sources, notably animal tissues (Provecho et al., 1984), bacteria including Lactococcus (formerly Streptococcus) lactis (Crow, 1990; Arora et al., 1978), Bacillus species and Enterbacter species (Giovannini et al. 1996), and yeast (Gibson et al., 1991). Boumerdassi et al. 1997 disclosed a mutated Lactococcus latis strain having DR activity that was increased by three times relative to the activity of the parent strain. In Arora et al. 1978 and Kulia and Rangenathan 1978 are disclosed mutants of Lactococcus lactis having a reduced diacetyl activity when grown in non fat dry milk and citrate medium, respectively.
Generally, the term xe2x80x9cdiscetyl reductasexe2x80x9d (xe2x80x9cDRxe2x80x9d) encompasses several enzymatic activities such as diacetyl reductase activity, acetoin reductese activity and/or butanediol dehydrogenase activity which carry out the following enzymatic reactions; diacetyl+NAD(P)H--- greater than acetoin+NAD(P)+, acetoin+NAD(P)H less than --- greater than butanediol+NAD(P)+, respectively. Thus, L. lactis has been reported to possess two diacetyl reductases with activity for both diacetyl and acetoin. Both of these enzymes use NADH as cofactor (Crow, 1990).
Leuconostoc species including Leu. pseudomesenteroides are typically used in mixed starter cultures together with Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. lactis biovar. diacetylactis in the production of dairy products. A significant role of Leuconostoc species in such mixed cultures is to remove the acetaldehyde produced by the accompanying strains e.g. in the production of buttermilk and fresh cheeses. However, Leuconostoc strains will also remove diacetyl by reducing it into acetoin and/or butanediol, a characteristic that is generally undesirable in the production of dairy products. The enzyme responsible for the reduction of diacetyl, diacetyl reductase, is highly expressed in Leuconostoc species such as Leu. psoudomesenteriodes which species is known to have about 100 times higher diacetyl reductese activity than L. lactis. 
Thus, one primary objective of the present invention is to provide lactic acid bacterial cultures of species, including Leuconastoc species, that inherently have one or more DR activities which, relative to the naturally occurring strains, has reduced or substantially eliminated DR activities under specific cofactor conditions. By providing such strains to the industry, it has become possible to produce lactic acid bacterial fermented food products having a desirably high content of diacetyl.
Another objective of the invention is to provide lactic acid bacterial strain that, relative to the presently available strains, has a strongly enhanced DR activities. Using such strains which utilise diacetyl as a substrate it is possible to reduce or remove diacetyl in food products where the presence of this flavour compound is undesirable.
Accordingly, the invention provides in a first aspect a genetically modified lactic acid bacterium, including the Leuconostoc pseudomesenteroides strains DSM 12099 and DSM 12465 and lactic acid bacteria essentially having the diacetyl reductase characteristics of these strains, that, relative to the lactic acid bacterium from which it is derived, is modified to have a reduction of at least one of diacetyl reductase activity, acetoin reductase activity and butanediol dehydrogenase activity, said modified bacterium,
(i) is substantially incapable of at least one of diacetyl reductase activity and acetoin reductase activity in a medium containing NADH and not containing NADPH, or
(ii) is substantially incapable of at least one of diacetyl reductase activity and acetoin reductase activity in a medium containing NADPH and not containing NADH, or
(iii) is substantially incapable of at least one of diacetyl reductase activity and acetoin reductase activity in a medium containing both NADH and NADPH, or
(iv) is substantially incapable of butanediol dehydrogenase activity in a medium containing NAD+ and not containing NADP+, or
(v) is substantially incapable of butanediol dehydrogenase activity in a medium containing NADP+ and not containing NAD+, or
(vi) is substantially incapable of butanediol dehydrogenase activity in a medium containing both NAD+ and NADP+,
where the bacterium prior to being modified is capable of having at least one of said enzymatic activities under said cofactor conditions.
In a further aspect, the invention relates to a genetically modified lactic acid bacterium that, relative to the lactic acid bacterium from which it is derived, is modified to have a reduction of at least one of diacetyl reductase activity, acetoin reductase activity and butanediol dehydrogenase activity, including the Leuconostoc pseudomesenteroides strains DSM 12099 and DSM 12465 and lactic acid bacteria essentially having the diacetyl reductase characteristics of these strains, subject to the limitation, that the lactic acid bacterium is not Lactococcus lactis. 
In a still further aspect, the invention relates to a genetically modified lactic acid bacterium that has no detectable diacetyl reductase activity, acetoin reductase activity and/or butanediol dehydrogenase activity, subject to the limitation, that the lactic acid bacterium is not Lactococcus lactis. 
In other further aspects, the invention relates to a genetically modified lactic acid bacterium that, relative to the lactic acid bacterium from which it is derived, is modified to have an enhancement of at least one of diacetyl reductase activity, acetoin reductase activity and butanediol dehydrogenase activity which is at least 10 times, including the Lactococcus lactis subsp, lactis strain DSM 12096 and lactic acid bacteria essentially having the diacetyl reductase characteristics of that strain.
In a still further aspect, the invention pertains to a starter culture composition comprising such a genetically modified bacterium.
There is also provided a method of preparing a fermented food product, comprising adding an effective amount of a bacterium that, relative to the lactic acid bacterium from which it is derived, is modified to have a reduction of at least one of diacetyl reductase activity, acetoin reductase activity and butanediol dehydrogenase activity, or a composition comprising such a bacterium to a food product starting material wherein the bacterium or the composition is incapable of having at least one enzymatic activity selected from the group consisting of diacetyl reductase activity, acetoin reductase activity and butanediol dehydrogenase activity and keeping the starting material under conditions where the bacterium or the starter culture composition is capable of fermenting said starting material to obtain the fermented food, and a fermented food product obtainable by such a method which product has a content of discetyl which is at least 10% higher than that of a product fermented under identical conditions with a parent strain for the genetically modified bacterium.
In yet another aspect, the invention relates to a method of producing a food product, comprising adding an effective amount of a bacterium that, relative to the lactic acid bacterium from which it is derived, is modified to have an enhancement of at least one of diacetyl reductase activity, acetoin reductase activity and butanediol dehydrogenase activity, or a composition comprising such a bacterium to a food product starting material that contains at least one of diacetyl, acetoin and butanediol, and keeping the starting material under conditions where the genetically modified lactic acid bacterium has at least one enzymatic activity selected from the group consisting of diacetyl reductase activity, acetoin reductase activity and butanediol dehydrogenase activity to obtain a product having a reduced content of diacetyl.
It is, as it is mentioned above, an important objective of the present invention to provide lactic acid bacteria that has a reduced capability to convert diacetyl in a fermented food product to acetoin and/or butanediol. Accordingly, in one aspect the genetically modified lactic acid bacterium is a bacterium that, relative to the lactic acid bacterium from which it is derived, is modified so as to have a reduction of at least one of diacetyl reductase activity, acetoin reductase activity and butanediol dehydrogenase activity, said bacterium, when grown under at least one of the above cofactor conditions, where the bacterium prior to being mutated is capable of having at least one of said enzymatic activities, is substantially incapable of at least one of said activities. As used herein, the term xe2x80x9csubstantially incapablexe2x80x9d indicates that the respective enzymatic activities can not be detected by the assay procedures described herein.
As used herein, the expression xe2x80x9clactic acid bacteriumxe2x80x9d refers to a group of gram-positive, microaerophilic or anaerobic bacteria having in common the ability to ferment sugars and citrate with the production of acids including lactic acid as the predominantly produced acid, acetic acid, formic acid and propionic acid. The industrially most useful lactic acid bacteria are found among Lactococcus species, Streptococcus species, Lactobacillus species, Leuconostoc species, Oenococcus species and Pediococcus species. In the dairy industry, the strict anaerobes belonging to the genus Bifidobacterium is generally included in the group of lactic acid bacteria as these organisms also produce lactic acid and are used as starter cultures in the production of dairy products.
It will be appreciated that the term xe2x80x9cgenetically modifiedxe2x80x9d as used herein indicates any modification of DNA sequences coding for genes involved in the expression of DR activities including modifications of sequences that regulate the expression of genes coding for such enzymatic activities. Accordingly, genetic modification can be based on construction or selection of mutants of lactic acid bacteria or it can be based on recombinant DNA-technology. When the term xe2x80x9cdiacetyl reductasexe2x80x9d or xe2x80x9cDRxe2x80x9d is used herein it refers to any of the three mentioned specific activities, i.e. diacetyl reductase activity, acetoin reductase activity and butanediol dehydrogenase activity.
As used herein the term xe2x80x9cmutantxe2x80x9d is used in the conventional meaning of that term i.e. it refers to strains obtained by subjecting a lactic acid bacterial strain to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethanemethane sulphonate (EMS) or N-methyl-Nxe2x80x2-nitro-N-nitroguanidine (NTG), UV light or to spontaneously occurring mutants which are selected on the basis of a modified DR activity. Although it is presently preferred to provide the genetically modified bacteria according to the invention by random mutagenesis or by selection of spontaneously occurring mutants, i.e. without the use of recombinant DNA-technology, it is envisaged that mutants of lactic acid bacteria can be provided by such technology including site-directed mutagenesis and PCR techniques and other in vitro or in vivo modifications of DNA sequences coding for DR activities or sequences regulating the expression of genes coding for the DR activities, once such sequences have been identified and isolated.
It is also possible to construct genetically modified bacteria according to the invention by conventional recombinant DNA-technology including insertion of sequences coding for DR activities, e.g. by replacing a native promoter for such coding sequences by a foreign promoter which either enhances or reduces the expression of the coding sequences. It is also possible to derive lactic acid bacterial strains according to the invention from species that do not have an inherent capability to produce DR activities by inserting genes coding for such activities isolated from a different organism comprising such genes. The source of such genes may be bacterial species, yeast species or mammal species. Additionally, it is envisaged that genetically modified bacteria according to the invention can be constructed by modifying metabolic pathways in a lactic acid bacterium that are not directly involved in DR pathways. It will be appreciated that the expression xe2x80x9cunder cofactor conditionsxe2x80x9d as used herein indicates the absence/presence in an appropriate medium of any non-protein substance required for biological activity of any of the enzyme activities according to the invention, such as NAD+, NADH, NADP+ and NADPH.
A genetically modified bacterium having a reduced diacetyl activity can be selected from any kind of lactic acid bacterial species which has an inherent DR activity, including Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp such as Leuconostoc pseudomesenteroides, Pediococcus spp., Oenococcus spp, and Bifidobacterium spp.
As mentioned above, the invention relates in another aspect to a genetically modified lactic acid bacterium that, relative to the lactic acid bacterium from which it is derived, is modified to have a reduction of at least one of diacetyl reductase activity, acetoin reductase activity and butanediol dehydrogenase activity, including the Leuconostoc pseudomesenteroides strains DSM 12099 and DSM 12465 and lactic acid bacteria essentially having the diacetyl reductase characteristics of these strains, subject to the limitation that the modified bacterium is not Lactococcus lactis. 
However, in particularly useful embodiments, the above genetically modified bacterium is one that under cofactor conditions, where the bacterium prior to being genetically modified is capable of having at least one of diacetyl reductase activity, acetoin reductese activity and butanediol dehydrogenase activity, is substantially incapable of at least one of said enzymatic activities.
Such a bacterium includes a bacterium that is substantially incapable of at least one of diacetyl reductase activity and acetoin reductase activity in a medium containing NADH and not containing NADPH, a bacterium that is substantially incapable of at least one of diacetyl reductase activity and acetoin reductase activity in a medium containing NADPH and not containing NADH, a bacterium that is substantially incapable of at least one of diacetyl reductase activity and acetoin reductase activity in a medium containing both NADH and NADPH, a bacterium that is substantially incapable of butanediol dehydrogenase activity in a medium containing NAD+ and not containing NADP+, a bacterium that is substantially incapable of butanediol dehydrogenase activity in a medium containing NADP+ and not containing NAD+ and a bacterium that is substantially incapable of butanediol dehydrogenase activity in a medium containing both NAD+ and NADP+.
In a further aspect, the invention provides a genetically modified lactic acid bacterium that has no detectable diacetyl reductase activity, acetoin reductase activity and/or butanediol dehydrogenase activity. Such a bacterium is selected from any of the above-mentioned lactic acid bacterial species, subject to the limitation, that the bacterium is not Lactococcus lactis. 
A genetically modified bacterium having reduced or no detectable DR activities can be derived from any lactic acid bacterial species which has an inherent DR activity, including Lactococcus spp. such as Lactococcus lactis subsp. lactis biovar. diacetylactis and Lactococcus lactis subsp. lactis, Streptococcus spp. including Streptococcus thermophilus, Lactobacillus spp., Leuconostoc spp. including Leuconostoc pseudomesenteroides., Pediococcus spp., Oenococcus spp. and Bifidobacterium spp.
Although it may be preferred that the modified bacterium has substantially no detectable DR activities, a bacterium that is modified to have a reduction of one or more of the above activities is also encompassed by the invention Thus, a useful bacterium according to the invention is one that has a reduction in any of the DR activities which, relative to the bacterium from which it is derived, is at least 25% such as at least 50% including at least 75% e.g. at least 90%. Thus, the genetically modified bacterium according to the invention preferably has a DR activity reduction which is reduced by at least 25 times for anyone of the enzymatic activities such as at least 50 times, including at least 100 times or even at least 500 times, relative to the strain from which it is derived.
When a modified lactic acid bacterial strain according to the invention is added to a food product starting material, such as e.g. milk, wherein the bacterium is incapable of having at least one of the above DR enzymatic activities and the starting material is kept under conditions where the strain is capable of fermenting said starting material to obtain a fermented food product, the resulting food product preferably has an increased content of diacetyl which is at least 1.1 times higher, such as at least 2 times higher, including at least 5 times higher or even at least 10 times higher, relative to a similar food product which is fermented using the strain from which the modified strain is derived.
Thus in one embodiment, the modified bacterium according to the invention is derived by subjecting a parent lactic acid bacterial strain that under appropriate cofactor conditions is capable of having diacetyl reductase activity, acetoin reductase activity and/or butanediol dehydrogenase activity to a mutagenization treatment and selecting a strain that is substantially incapable of at least one of said enzymatic activities under identical cofactor conditions.
The present invention relates in a further aspect to a genetically modified lactic acid bacterium that, relative to the lactic acid bacterium from which it is derived, is modified to have an enhancement of at least one of diacetyl reductase activity, acetoin reductase activity and butanediol dehydrogenase activity which is at least 10 times, including the Lactococcus lactis subsp. lactis strain DSM 12096 and lactic acid bacteria essentially having the diacetyl reductase characteristics of that strain.
It was found that it is possible to provide genetically modified lactic acid bacteria that have a significant enhancement of the specific DR activities. Thus, by fermenting a material or a medium having a content of diacetyl with such a genetically modified bacterium it is possible to obtain a final product wherein essentially all of the diacetyl has been converted to butanediol which is without the buttery flavour of diacetyl. Thus, the genetically modified bacterium according to the invention preferably has an activity enhancement which is at least 10 times for anyone of the enzymatic activities such as at least 50 times or even at least 100 times, relative to the strain from which it is derived.
A genetically modified bacterium having enhanced DR activities can be derived from any lactic acid bacterial species which has an inherent DR activity, including Lactococcus spp. such as Lactococcus lactis subsp. lactis biovar. diacetylactis and Lactococcus lactis subsp. lactis, Streptococcus spp. including Streptococcus thermophilus, Lactobacillus spp., Leuconostoc spp. including Leuconostoc pseudomesenteroides., Pediococcus spp. and Bifidobacterium spp.
It will be appreciated that such modified bacteria can be a spontaneous mutant or be provided by subjecting a lactic acid bacterium that has inherent DR activities to a mutagenization treatment as described above or by inactivating or deleting one or more genes involved in the expression of the DR activities using conventional recombinant DNA-technology.
The genetically modified bacteria according to the invention are useful as starter cultures in the production of food products. Accordingly, in a further important aspect, the invention relates to a starter culture composition comprising a bacterium according to the invention either having enhanced or a reduced or eliminated DR activities.
Typically, such a composition comprises the bacteria in a concentrated form including frozen, dried or freeze-dried concentrates typically having a concentration of viable cells which is in the range of 104 to 1012 cfu per g including at least 104 cfu per gram of the composition, such as at least 105 cfu/g, e.g. at least 106 cfu/g, such as at least 107 cfu/g, e.g. at least 108 cfu/g, such as at least 109 cfu/g, e g. at least 1010 cfu/g, such as at least 1011 cfu/g of the composition. The composition may as further components contain cryoprotectants and/or conventional additives including nutrients such as yeast extract, sugars and vitamins.
As it is normal in the production of lactic acid bacterial fermentation processes to apply mixed cultures lactic acid bacteria, the composition will in certain embodiments comprise a multiplicity of strains either belonging to the same species or belonging to different species. A typical example of such a useful combination of lactic acid bacteria in a starter culture composition is a mixture of a Leuconostoc spp, and one or more Lactococcus spp. such as Lactococcus lactis subsp. lactis or Lactococcus lactis subsp. lactis biovar. diacetylactis. Such a mixed culture can be used in the manufacturing of fermented milk products such as buttermilk and cheese. It will be understood that in such a mixed culture of lactic acid bacteria, one or more of the strain components may be a modified bacterium according to the invention.
It is also an objective of the invention to provide a method of preparing a fermented food product based on the use of the genetically modified bacteria of the invention which have reduced or lacking DR activities. In its broadest aspect, such a method comprises that an effective amount of such bacteria or a composition comprising the bacteria are added to a food product starting material wherein the bacterium or the composition is incapable of having at least one of the above DR enzymatic activities and keeping the starting material under conditions where the bacterium or the culture composition is capable of fermenting said starting material to obtain a fermented food product.
Useful food product starting materials include any material which is conventionally subjected to a lactic acid bacterial fermentation step such as milk, vegetable materials, meat products, fruit juices, must, doughs and batters. The fermented products which are obtained by the method include as typical examples dairy products such as cheese including fresh cheese products, and buttermilk.
As it is mentioned above, the use in food starter cultures of bacteria according to the invention that have a reduced or lacking DR activity will result in final products having a content of the desired flavour compound diacetyl which is higher than would otherwise be obtained if a non-modified lactic acid bacterium was used. Accordingly, it is an important aspect of the invention to provide a fermented food product obtainable by the above method which product has a content of diacetyl which is at least 10% higher such as at least 20% higher or at least 30% higher than that of a product fermented under identical conditions with a parent strain for the genetically modified bacterium Examples of such food products include milk-based products such as cheese and buttermilk, vegetable products, meat products, fruit juices, wines and bakery products.
As shown in the below Examples, when the DR mutant MM084 is used as a component of a mixed flavour-forming starter culture for the fermentation of one of the above starting materials, the mutant has a significant effect on the diacetyl stability during storage of the resulting fermented product.
Thus, an advantageous feature of the fermented food product according to the invention is that the food product can be stored for several weeks with less reduction in the diacetyl content than is the case with a food product fermented under identical conditions with the parent strain of the genetically modified bacterium. Thus, in one particularly useful embodiment, the fermented product is one which at least 10% of its initial diacetyl content is retained after storage for 20 days of more at about 4xc2x0 C. when stored under appropriate storage conditions, such as at least about 20% of its initial diacetyl content e.g. at least about 30% and preferably at least about 40% e.g. at least about 50% of its initial diacetyl content is retained after storage for 20 days or more at about 4xc2x0 C. This improvement implies that a fermented food product manufactured by use of the above mixed starter culture can be stored for an extended period of time without loosing its desired flavour.
Whereas in many lactic acid bacterial fermented food products it is desirable to have a high content of diacetyl, this may be undesirable in other products. This is in particular the case in beverages such as fruit juices, beers and other yeast fermented beverages including certain wines, where diacetyl imparts to the products a buttery or toffee taste. In beers a diacetyl content above the threshold level gives rise to the so-called sarcina sickness. It is therefore an interesting aspect of the invention to provide a method of producing a food product having a reduced content of diacetyl.
This method comprises adding an effective amount of a lactic acid bacterium that has been modified to have at least one increased DR activity or a composition containing such a bacterium to a food product starting material that contains at least one of diacetyl, acetoin and butanediol, and keeping the starting material under conditions where the genetically modified lactic acid bacterium has at least one enzymatic activity selected from the group consisting of diacetyl reductase activity, acetoin reductase activity and butanediol dehydrogenase activity to obtain a product having a reduced content of diacetyl. In useful embodiments the products resulting from such a method have no detectable content of diacetyl.